Sample Injector With Metering Device Balancing Pressure Differences In An Intermediate Valve State

ABSTRACT

A sample injector for use in a fluid separation system for separating compounds of a fluidic sample in a mobile phase, the sample injector comprising a switchable valve, a sample loop in fluid communication with the valve and configured for receiving the fluidic sample, a metering device in fluid communication with the sample loop and configured for introducing a metered amount of the fluidic sample on the sample loop, and a control unit configured for controlling switching of the valve to transfer the sample loop between a low pressure state and a high pressure state via an intermediate state and for controlling the metering device during the intermediate state to at least partially equilibrate a pressure difference in the sample loop between the low pressure state and the high pressure state.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application under 37 C.F.R. §1.53(b) of commonly owned U.S. patent application Ser. No. 16/993,070,filed on Aug. 13, 2020; which is a continuation of U.S. patentapplication Ser. No. 15/965,402, filed on Apr. 27, 2018; which is acontinuation application under 37 C.F.R. § 1.53(b) of commonly ownedU.S. patent application Ser. No. 15/622,913, filed on Jun. 14, 2017;which is a continuation application under 37 C.F.R. § 1.53(b) ofcommonly owned U.S. patent application Ser. No. 14/877,758, filed onOct. 7, 2015; which is a continuation application under 37 C.F.R. §1.53(b) of commonly owned U.S. patent application Ser. No. 13/375,884,filed on Dec. 2, 2011; which is the national stage application ofInternational Application No. PCT/EP2009/056795, filed on Jun. 3, 2009;the contents of each of which are incorporated by reference herein intheir entireties.

BACKGROUND ART

The present invention relates to sample injectors, in particular in ahigh performance liquid chromatography application.

In high performance liquid chromatography (HPLC, see for instancehttp://en.wikipedia.org/wiki/HPLC), a liquid has to be provided usuallyat a very controlled flow rate (e.g. in the range of microliters tomilliliters per minute) and at high pressure (typically 20-100 MPa,200-1000 bar, and beyond up to currently 200 MPa, 2000 bar) at whichcompressibility of the liquid becomes noticeable. For liquid separationin an HPLC system, a mobile phase comprising a sample fluid withcompounds to be separated is driven through a stationary phase (such asa chromatographic column), thus separating different compounds of thesample fluid.

Valves are commonly used in HPLC applications, for instance injectionvalves for introducing a liquid sample into a high pressure flowingstream of liquid, a purge valves for positive displacement pumps, flowpath switching valves, etc. Such valves used in HPLC applications areoften multi-position rotary valve. Examples of multi-position rotaryvalve are disclosed in U.S. Pat. No. 4,068,528 A (two-position valves)or US 2003/0098076 A1 (multi-function rotary valves or random-access,dual, three-way, rotary switching valves).

Shear valves, which can be used in multi-way embodiments, are usuallyformed by a housing and a body defining a stepped cavity in which therotor or seal is positioned. The housing contains at least two shearseal valve members positioned to be aligned with ports in the rotor(body) to establish communication between the shear seal means. Shearvalves are usually provided as rotary valves (such as the aforementionedrotary valves) or translational valves (often also called slidingvalves), such as disclosed in EP 0321774 A2.

A multi-way switching valve provides a means for selectively routing afluid input flow to the valve to one of more alternate output flows fromthe valve. A rotary valve is of the type wherein fluid flow is directedby rotating a valve rotor element to discrete angular positions relativeto a stationary valve stator element. A dual rotary valve provides twovalves in one valve body, both simultaneously operated by thepositioning of the valve rotor. Rotary switching valves are commonlyused, for example, in HPLC and other analytical methods to selectivelydirect a flow stream of one or more fluids along alternate paths to ananalytical device or containment vessel.

The aforementioned US 2003/0098076 A1 shows in its FIG. 1 a conventionaltype of dual, three-way, switching valve 220, which includes adisc-shaped rotor with a set of rotor grooves in the front face of therotor that contacts, in a fluid-tight manner, the face of acylindrically shaped stator body at a rotor-stator interface. Inletpassages and outlet passages, longitudinally bored through the statorbody to the rotor-stator interface, are selectively fluidly coupledthrough the rotor grooves corresponding to the rotation of the rotorrelative to the stator. Pivoting of the rotor enables the rotor groovesto fluidly couple selected passages of the stator, depending on theirplacement on the rotor and the angular position of the valve rotor.Model 7030 of Rheodyne, L. P. is an example of this type of switchingvalve.

WO 2007/109529 discloses methods and apparatus for placing a sample in achromatographic system. The device and method feature placing samplesheld in a sample loop to pressurization prior to placing such sampleloop in communication with high pressure conduits.

WO 2008/005845 discloses a method for processing a fluid applied tosystems that include a valve unit that has a sample-loading state and asample-introducing state. The sample-loading state disposes a sampleloop in fluidic communication with a sample conduit. Thesample-introducing state disposes the sample loop in fluidiccommunication with a process conduit. The method involves transferring asample through both the sample conduit and the valve unit so that aleading end of the sample exits the valve unit. After transitioning thevalve unit to the sample-loading state and allowing the sample loop todecompress, at least some of the transferred sample is loaded into thesample loop. A fluid-processing instrument includes a value unit and acontrol unit that manages operation of the instrument. The control unitis configured, for example, to implement the above-described method.

WO 2006/083776 discloses a method and apparatus for substantiallyeliminating destructive transients of pressure or flow rate which candegrade the efficiency and useful lifetime of chromatography columns.The system enables a substantially constant flow of mobile phase liquidto be maintained through the chromatography system by eliminating theflow blockage interval associated with the actuation of sample injectionvalves. The system further provides a method to reduce the pressure andflow rate transients associated with pressurization of the sample loopcontents when the sample loop is introduced to chromatography systemdelivery pressure.

WO 2006/023828 discloses systems, devices, and methods to mitigate thepressure disturbance associated with the injection of low-pressureanalyte samples into a high-pressure HPLC fluid stream, to enhancechromatographic performance related to retention time andreproducibility. An embodiment coordinates the injection run with activepressure control of a binary solvent delivery system to virtuallyeliminate the customary pressure drop when the low-pressure loop isbrought on line. An additional feature is accomplished by forcing aconsistent timing relationship between the injection run, the mechanicalposition of the delivery pump pistons, and the start and subsequentgradient delivery.

US 2007/0251302 discloses a flow path switching valve in which an impactdue to the pressure change when a flow path is switched is preventedfrom being generated. A rotor slot allows an analysis infusion pump tobe connected to an analytical column, so as to form a flow path(condensing procedure). The rotor of the flow path switching valve isrotated clockwise for 30 degrees, the rotor slot allows the analysisinfusion pump, the analytical column, and a trap column be connected.After the pressure in the trap column is raised to the same pressurelevel as that of the analytical column, the pressure is stabilized, andthe pressure difference between the two columns is counteracted(high-pressure procedure). After the pressure between the two columnshas been stabilized sufficiently, the rotor is further rotated for 30degrees, and the trap column and the analytical column are connected inseries, so the sample analysis can be performed (dissolution procedureand detection procedure).

In modern HPLC with pressures rising up to 100 MPa and beyond, life timeof sample injectors becomes critical, in particular for the injectionvalve, as a high pressure load acts on the components particularly whenswitching between a high pressure operation mode and a low pressureoperation mode, which causes excessive wear.

DISCLOSURE

It is an object of the invention to provide an improved sample injector,in particular for high pressure HPLC applications. The object is solvedby the independent claims. Further embodiments are shown by thedependent claims.

According to an embodiment of the present invention, a sample injectorfor use in a fluid separation system for separating compounds of afluidic sample in a mobile phase is provided, the sample injectorcomprising a switchable valve (i.e. a valve switchable between multiplepositions, wherein each position corresponds to an assigned fluidcoupling/decoupling characteristic of conduits connectable to thevalve), a sample loop in fluid communication with the valve andconfigured for receiving the fluidic sample, a metering device in fluidcommunication with the sample loop and configured for introducing ametered amount (for instance a predefined volume or mass) of the fluidicsample on the sample loop, and a control unit configured for controllingswitching of the valve to transfer the sample loop between a lowpressure state (at which the sample loop may be at a first pressurevalue) and a high pressure state (at which the sample loop may be at asecond pressure value larger than the first pressure value) via anintermediate state and for controlling the metering device during theintermediate state to at least partially (i.e. partially or entirely)equilibrate a pressure difference in the sample loop between the lowpressure state and the high pressure state.

According to another embodiment of the present invention, a fluidseparation system for separating compounds of a fluidic sample in amobile phase is provided, the fluid separation system comprising amobile phase drive (such as a pumping system) adapted to drive themobile phase through the fluid separation system, a separation unit(such as a chromatographic column) adapted for separating compounds ofthe fluidic sample in the mobile phase, and a sample injector having theabove mentioned features for introducing the fluidic sample into themobile phase.

According to still another embodiment of the present invention, a methodof operating a sample injector in a fluid separation system forseparating compounds of a fluidic sample in a mobile phase is provided,wherein the method comprises introducing, by a metering device, ametered amount of the fluidic sample on a sample loop in fluidcommunication with a switchable valve and the metering device,controlling switching of the valve to transfer the sample loop between alow pressure state and a high pressure state via an intermediate state,and controlling the metering device during the intermediate state to atleast partially equilibrate a pressure difference in the sample loopbetween the low pressure state and the high pressure state.

According to an exemplary embodiment, a switchable valve, a sample loopand a metering device may be arranged in a configuration in which theyare always in fluid communication with one another regardless of apresent switching state of the valve. The system may be switchablebetween two or more pressure modes, particularly between a high pressuremode and a low pressure mode. Exemplary embodiments may allow tosuppress or even eliminate undesired pressure drops and consequentlyundesired cavitation effects (such as bubble implosions in theswitchable valve or the sample loop in response to a sudden change ofthe pressure conditions) by softly equilibrating the pressure differencebetween the two pressure modes in a dedicated intermediate valve stateso that a smooth balancing of pressure differences between the lowpressure state and the high pressure state can be achieved. Cavitationeffects may deteriorate or even delaminate a coating of the valve. Bypreventing cavitation effects, the lifetime of the sample injector andparticularly of the switchable valve may be significantly increased.Furthermore, a sudden pressure increase or decrease may result indisturbances in a flow profile, and may interrupt a column flow duringthe switching procedure. Also such undesired effects may be efficientlysuppressed by exemplary embodiments.

In the following, further exemplary embodiments of the sample injectorwill be explained. However, these embodiments also apply to the fluidseparation system and to the method.

According to an exemplary embodiment, the control unit may be configuredfor controlling switching of the valve to transfer the sample loop fromthe high pressure state to the low pressure state via the intermediatestate and for controlling the metering device during the intermediatestate to perform a decompression (or pressure reduction) in the sampleloop before transferring the sample loop to the low pressure state. Insuch an embodiment, the smooth equilibration starts in a high pressurestate in which a high pressure of for instance 100 MPa is present at thesample loop and decompresses the sample loop for reducing the pressuretowards or up to a low pressure (for instance an atmospheric pressure)before initiating the switching to the actual low pressure state. Thismay safely prevent sudden decompression of a fluid which may occur inthe sample loop when switching from the high pressure to the lowpressure.

Still referring to the previous embodiment, the control unit may beconfigured for controlling the metering device for performing thedecompression by retracting a metering piston of the metering device.Hence, the metering device which is present in the sample injectorpredominantly for introducing a sample from a vial or the like into thesample loop by retracting and forwarding a metering piston, may be usedas well for performing the decompression prior to the switching. Hence,the metering device can be synergistically used for both purposes ofsample introduction and pressure equilibration.

Additionally or alternatively, the control unit may be configured forcontrolling switching of the valve to transfer the sample loop from thelow pressure state to the high pressure state via the intermediate stateand for controlling the metering device during the intermediate state toperform a precompression in the sample loop before transferring thesample loop to the high pressure state. Hence, the pressureequilibration feature can be applied also in a configuration in which aswitch from the low pressure mode to the high pressure is initiated sothat in the intermediate valve state the pressure may be slowly orcontinuously increased so that a subsequent switch from the intermediatestate to the high pressure state of the valve does not generate anintense pressure pulse since the pressure difference has already beenequilibrated smoothly beforehand.

Still referring to the previous embodiment, the control unit may beconfigured for controlling the metering device for performing theprecompression by pushing forward the metering piston. As mentionedabove, the metering device may predominantly act for introducing asample from a vial into the sample loop but may be, according to thedescribed exemplary embodiment, used as well for effecting a pressureincrease in the sample loop during the intermediate state for reducing amechanical load which conventionally acts on the components of thesample injector upon suddenly switching from the low pressure mode tothe high pressure mode.

In an embodiment, the metering device may be configured as a highpressure metering device. In other words, the metering device may beconfigured for providing pressure values which are significantly higherthan only several bars, thereby providing the structural and functionalcapability of equilibrating the pressure in the sample loop between highand low pressure modes with typical pressure values which can be presentin the sample injector of a liquid chromatography device such as a HPLC.This may require to substitute conventional metering devices (likesyringe pumps, capable of operating at a pressure value of several barsonly) by a high pressure metering device which may be capable ofproviding significantly larger pressures such as about 10 MPa,particularly at least about 50 MPa, more particularly at least about 100MPa or more.

The metering device may be configured for providing basically the samepressure as a mobile phase drive, particularly a pumping system, adaptedto drive a mobile phase through a separation column of the fluidseparation system. Such a mobile phase drive may be provided to drive amobile phase such as a solvent composition comprising, for instance, amixture of water and an organic solvent such as ACN, for conducting thesame through a separation column of a liquid chromatography device. Thementioned metering device may provide a pressure of for instance 100MPa, whereas sample introduction into the sample loop using a meteringdevice is in many conventional cases performed not significantly abovean atmospheric pressure or the like. Hence, the metering device may thenbe operated in different pressure modes, for instance a low pressuremode for introducing a sample from a vial or the like into the sampleloop or in a high pressure state for bringing the sample loop smoothlyto a high pressure value as provided by the mobile phase drive beforeswitching a valve from an intermediate to a high pressure state.

The metering device may also be configured for increasing a pressure inthe sample loop, before switching the sample loop from the low pressurestate to the high pressure state, to or towards a system pressure of amobile phase drive, particularly a pumping system, adapted to drive amobile phase through a separation column of the fluid separation system.Hence, the metering device may dampen the pressure drop between a modein which the sample loop is in fluid communication with the mobile phasedrive and a mode in which the sample loop is out of fluid communicationwith the mobile phase drive.

According to an exemplary embodiment, the valve may comprise a firstvalve member and a second valve member, wherein at least one of thefirst and the second valve members is adapted to be moved with respectto the other, wherein one of the first and second valve memberscomprises a plurality of ports and the other comprises at least onegroove for fluidly coupling respective ones of the ports in dependencyon a relative movement position of the first and the second valvemembers with respect to each other. In other words, fluid paths may beformed by at least two of the ports and at least one of the grooveswhich can selectively be brought in or out of fluid communication withthese ports. In contrast to conventional approaches in which such avalve only has an initial state and a final state and a switchingbetween the initial state and the final state suddenly increases ordecreases a pressure, a valve according to an exemplary embodiment mayhave a stable intermediate state between the initial and the final stateto which intermediate state the valve may be brought for performingequilibration between a high pressure and a low pressure using themetering device in the sample loop.

Still referring to the previous embodiment, the plurality of ports andthe at least one groove may be designed so that in the intermediatestate of the sample loop succeeding the low pressure state and precedingthe high pressure state of the sample loop, a pumping system adapted todrive the mobile phase through a separation column is still in fluidcommunication with the separation column, and the sample loop is nolonger at an atmospheric pressure and not yet in fluid communicationwith the separation column. Such an embodiment corresponds to a switchfrom a bypass mode to a main pass mode (for instance to a switch fromFIG. 4 via FIG. 3 to FIG. 2 ). Therefore, in the intermediate state, thesample loop may still be fluidly decoupled from the pumping system butcan be already brought to a higher pressure as compared to 1 bar beforebeing switched to the start/inject state.

Additionally or alternatively, the plurality of ports and the at leastone groove may further be designed so that in the intermediate state ofthe sample loop succeeding the high pressure state and preceding the lowpressure state of the sample loop, a pumping system adapted to drive themobile phase through a separation column is still in fluid communicationwith the separation column, and the sample loop is no longer at apressure of the pumping system and not yet at an atmospheric pressure.Such an embodiment corresponds to a switch from a main pass mode to abypass mode (for instance to a switch from FIG. 2 via FIG. 3 to FIG. 4). Therefore, in the intermediate state, the sample loop may be alreadyfluidly decoupled from the pumping system and can be already brought toa lower pressure before being switched to the loading state.

The plurality of ports and the at least one groove may be designed sothat in the high pressure state, the sample loop is in fluidcommunication with a pumping system adapted to drive a mobile phasedrive through a separation column and is in fluid communication with theseparation column. Thus, the high pressure state may be characterized bya fluid communication between the mobile phase drive and the sampleloop.

The plurality of ports and the at least one groove may further bedesigned so that in the low pressure state, the sample loop is not influid communication with a pumping system adapted to drive a mobilephase through a separation column and is not in fluid communication withthe separation unit. Thus, the low pressure state can be characterizedby the absence of the high pressure of the mobile phase drive in thesample loop.

Moreover, the plurality of ports and the at least one groove may bedesigned so that a first position of one of the at least one groove isaligned with one of the plurality of ports in the low pressure state, asecond position of the one of the at least one groove is aligned withthe one of the plurality of ports in the high pressure state, and athird position (differing from the first and second positions) of theone of the at least one groove is aligned with the one of the pluralityof ports in the intermediate state. In such an embodiment, a stopposition of the one of the plurality of ports may be defined(particularly not only at the first and the second position but also) ata third position of the one of the at least one groove. Therefore, anintermediate valve state may be provided which represents a stateselectable by the control unit during which an equilibration between ahigh pressure and a low pressure may be performed within the sampleloop.

In an embodiment, different ones of the plurality of grooves may havedifferent lengths. Therefore, by length selection and also geometryselection (the grooves may have an arcuate partial circle likeappearance but can also have further geometrical features such as a hookor the like), additional design parameters for valve configuration areprovided which allow to properly define intermediate state, initialstate, end state and optionally further states of the valve.

Optionally, a pressure sensor may be arranged in the sample loop(particularly between the metering device and the switchable valve) formeasuring a pressure in the sample loop. The pressure sensor may providea measured pressure to the control unit as a feedback signal as a basisfor the controlling of at least one of the metering device and thevalve.

The low pressure may be smaller than the high pressure. For example, thelow pressure may be an atmospheric pressure (of about 0.1 MPa), whereasthe high pressure may be at least 50 MPa, more particularly at leastabout 100 MPa. With such pressure drops between atmospheric pressure and50 MPa or even 100 MPa, strong and destructive cavitation effects mayoccur without the pressure equilibration according to an exemplaryembodiment.

According to one embodiment, the valve may comprise six ports and twogrooves. In such a configuration which is shown in the embodiment ofFIG. 2 to FIG. 4 for example, the intermediate state may be arrangedbetween two other valve states.

In an alternative embodiment, which is illustrated in FIG. 5 to FIG. 9 ,the valve may comprise seven ports, three grooves and may be switchablebetween six (or more) positions. With such a configuration, the pressureequilibration feature may be further refined.

The low pressure state of the sample loop may correspond to an operationmode in which the fluidic sample is loaded onto the sample loop and themobile phase is driven by a mobile phase drive, particularly a pumpingsystem, through a separation column of the fluid separation system. Insuch an operation mode, a needle can be lifted out of a seat in thesample loop and may be immersed into a vial or the like for loading thesample on the sample loop, which may occur at a relatively low pressureof for instance one atmosphere.

In contrast to this, the high pressure state of the sample loop maycorrespond to an operation mode in which the fluidic sample is injectedfrom the sample loop to the separation column and is driven by a mobilephase drive, particularly a pumping system, to be loaded onto theseparation column of the fluid separation system. In such an embodiment,the sample which has previously been loaded in the sample loop may thenbe pumped onto a separation column using the high pressure of the mobilephase drive. Subsequently, the different fractions of the sample whichare then retained at fluid separation beads of the separation column maybe individually and separately be released from the separation column bya gradient run, i.e. by a variation of a solvent which may besubsequently pumped through the separation column by the mobile phasedrive.

Optionally, the sample injector may comprise a flush conduit configuredfor flushing at least a part of fluidic conduits of the sample injector.For example for cleaning or rinsing purposes, a flush loop may beprovided which allows to clean such fluidic conduits to preventcarryover or the like. Such a flush loop may be properly implemented inthe pressure equilibration system according to an exemplary embodiment.

In an embodiment, the metering device is arranged within the sampleloop. In other words, in the described embodiment metering device andsample loop may be always in fluid communication with one anotherregardless of a switching state of the valve. This architecture mayallow for a very simple equilibration of the pressure in the sample loopwhen transferring the sample loop between a high pressure mode and a lowpressure mode.

According to an exemplary embodiment, an appropriate groove design in avalve may allow to provide an additional intermediate position at whichthe pump may still be connected with the column and the split loop maybe not yet connected with the pump and may be no more connected with awaste (atmospheric pressure). When providing a high pressure capablemetering device in the split loop, i.e. a high pressure metering devicecapable of providing the same pressures as a column pump, it is possibleto balance out pressure differences before the switching. For instance,such a high pressure metering device may displace a volume of forexample 2 .mu.l to 10 .mu.l with a pressure of about 100 MPa. To achievesuch a performance, it is possible to position the high pressuremetering device (particularly a piston position thereof) in such amanner that the compression of 2 .mu.l to 10 .mu.l is possible.

In one embodiment, the shear valve is embodied as a rotary valve, withthe first and second shear valve members being rotably moveable withrespect to each other. In another embodiment, the shear valve isembodied as a translational valve, such as a slide valve, with the firstand second shear valve members being translationally moveable withrespect to each other.

In one embodiment, the shear valve further comprises a housing forhousing one of the first and second shear valve members, wherein thehousing is pre-stressed (pre-loaded) against the housed shear valvemember. This allows reducing breakage or fracture stress, which mayoccur in the housed shear valve member. The housing is preferablyattached to the housed shear valve member by using a shrinking processas known in the art.

In one embodiment, the fluid path of this shear valve comprises agroove. In one embodiment, one or more of the ports of the shear valvecomprise a through hole having an opening fluidly coupling with thefluid path dependent on the moving position. In one embodiment, whereinthe first shear valve member comprises a plurality of ports, the secondshear valve member comprises the at least one fluid path for fluidlycoupling respective ones of the port independency on a relative movementposition of the first and second shear valve member with respect to eachother. In a further embodiment, the second shear valve member is adaptedto be moved with respect to the first shear valve member. Preferably,the second shear valve member is provided as rotor or slider moving onthe first shear valve member, which is embodied as static member and notmoving. A drive might be provided for moving the shear valve member tobe moved. Alternatively or in addition, the shear valve member to bemoved might also be moved manually. A valve drive and control unit (e.g.gearbox+motor+encoder+central processing unit, CPU), might be providedfor controlling movement of the shear valve member to be moved.

The shear valve is preferably adapted to conduct a liquid in the atleast one fluid path at a high pressure at which compressibility of theliquid becomes noticeable, such as pressure in the range of 20-200 MPa,and particularly 50-120 MPa.

The shear valve can be a sample injection valve for introducing a liquidsample into a high pressure flowing stream of liquid, a high pressurepurge valve for a positive displacement pump, or a flow path switchingvalve for switching from one flow path to another flow path.

The shear valve might be embodied in an HPLC sample injector adapted tointroduce a sample fluid into a mobile phase. The mobile phase is to bedriven by a mobile phase drive through a separation unit for separatingcompounds of the sample fluid in the mobile phase. A sample loop isprovided for receiving the sample fluid. The shear valve is provided forswitching the sample loop between the mobile phase drive and theseparation unit for introducing the sample fluid into the mobile phase.

Embodiments of the present invention might be embodied based on mostconventionally available HPLC systems, such as the Agilent 1200 SeriesRapid Resolution LC system or the Agilent 1100 HPLC series (bothprovided by the applicant Agilent Technologies—see www.agilent.com—whichshall be incorporated herein by reference).

One embodiment comprises a pumping apparatus having a piston forreciprocation in a pump working chamber to compress liquid in the pumpworking chamber to a high pressure at which compressibility of theliquid becomes noticeable.

One embodiment comprises two pumping apparatuses coupled either in aserial or parallel manner. In the serial manner, as disclosed in EP309596 A1, an outlet of the first pumping apparatus is coupled to aninlet of the second pumping apparatus, and an outlet of the secondpumping apparatus provides an outlet of the pump. In the parallelmanner, an inlet of the first pumping apparatus is coupled to an inletof the second pumping apparatus, and an outlet of the first pumpingapparatus is coupled to an outlet of the second pumping apparatus, thusproviding an outlet of the pump. In either case, a liquid outlet of thefirst pumping apparatus is phase shifted, preferably essentially 180degrees, with respect to a liquid outlet of the second pumpingapparatus, so that only one pumping apparatus is supplying into thesystem while the other is intaking liquid (for instance from thesupply), thus allowing to provide a continuous flow at the output.However, it is clear that also both pumping apparatuses might beoperated in parallel (i.e. concurrently), at least during certaintransitional phases for instance to provide a smooth(er) transition ofthe pumping cycles between the pumping apparatuses. The phase shiftingmight be varied in order to compensate pulsation in the flow of liquidas resulting from the compressibility of the liquid. It is also known touse three piston pumps having about 120 degrees phase shift.

The separating device preferably comprises a chromatographic column (seefor instance http://en.wikipedia.org/wiki/Column chromatography)providing the stationary phase. The column might be a glass or steeltube (for instance with a diameter from 50 .mu.m to 5 mm and a length of1 cm to 1 m) or a microfluidic column (as disclosed for instance in EP1577012 or the Agilent 1200 Series HPLC-Chip/MS System provided by theapplicant Agilent Technologies, see for instancehttp://www.chem.agilent.com/Scripts/PDS.asp?|Page=38308). For example, aslurry can be prepared with a powder of the stationary phase and thenpoured and pressed into the column. The individual components areretained by the stationary phase differently and separate from eachother while they are propagating at different speeds through the columnwith the eluent. At the end of the column they elute one at a time.During the entire chromatography process the eluent might be alsocollected in a series of fractions. The stationary phase or adsorbent incolumn chromatography usually is a solid material. The most commonstationary phase for column chromatography is silica gel, followed byalumina. Cellulose powder has often been used in the past. Also possibleare ion exchange chromatography, reversed-phase chromatography (RP),affinity chromatography or expanded bed adsorption (EBA). The stationaryphases are usually finely ground powders or gels and/or are microporousfor an increased surface, though in EBA a fluidized bed is used.

The mobile phase (or eluent) can be either a pure solvent or a mixtureof different solvents. It can be chosen for instance to minimize theretention of the compounds of interest and/or the amount of mobile phaseto run the chromatography. The mobile phase can also been chosen so thatthe different compounds can be separated effectively. The mobile phasemight comprise an organic solvent like for instance methanol oracetonitrile, often diluted with water. For gradient operation water andorganic is delivered in separate bottles, from which the gradient pumpdelivers a programmed blend to the system. Other commonly used solventsmay be isopropanol, THF, hexane, ethanol and/or any combination thereofor any combination of these with aforementioned solvents.

The sample fluid might comprise any type of process liquid, naturalsample like juice, body fluids like plasma or it may be the result of areaction like from a fermentation broth.

The pressure in the mobile phase might range from 2-200 MPa (20 to 2000bar), in particular 10-150 MPa (100 to 1500 bar), and more particular50-120 MPa (500 to 1200 bar).

The HPLC system might further comprise a sampling unit for introducingthe sample fluid into the mobile phase stream, a detector for detectingseparated compounds of the sample fluid, a fractionating unit foroutputting separated compounds of the sample fluid, or any combinationthereof. Further details of HPLC system are disclosed with respect tothe Agilent 1200 Series Rapid Resolution LC system or the Agilent 1100HPLC series, both provided by the applicant Agilent Technologies, underwww.agilent.com which shall be in cooperated herein by reference.

Embodiments of the invention can be partly or entirely embodied orsupported by one or more suitable software programs, which can be storedon or otherwise provided by any kind of data carrier, and which might beexecuted in or by any suitable data processing unit. Software programsor routines can be preferably applied in or by the control unit.

BRIEF DESCRIPTION OF DRAWINGS

Other objects and many of the attendant advantages of embodiments of thepresent invention will be readily appreciated and become betterunderstood by reference to the following more detailed description ofembodiments in connection with the accompanied drawing(s). Features thatare substantially or functionally equal or similar will be referred toby the same reference sign(s).

FIG. 1 shows a liquid separation system, in accordance with embodimentsof the present invention, for instance used in high performance liquidchromatography (HPLC).

FIG. 2 to FIG. 4 shows an exemplary embodiment of a sample injectoraccording to the present invention in different operation modes.

FIG. 5 to FIG. 9 shows another exemplary embodiment of a sample injectoraccording to the present invention in different operation modes.

Referring now in greater detail to the drawings, FIG. 1 depicts ageneral schematic of a liquid separation system 10. A pump 20 receives amobile phase from a solvent supply 25, typically via a degasser 27,which degases and thus reduces the amount of dissolved gases in themobile phase. The pump 20—as a mobile phase drive—drives the mobilephase through a separating device 30 (such as a chromatographic column)comprising a stationary phase. A sampling unit 40 (compare the detaileddescription of FIG. 2 to FIG. 9 ) can be provided between the pump 20and the separating device 30 in order to subject or add (often referredto as sample introduction) a sample fluid into the mobile phase. Thestationary phase of the separating device 30 is adapted for separatingcompounds of the sample liquid. A detector 50 is provided for detectingseparated compounds of the sample fluid. A fractionating unit 60 can beprovided for outputting separated compounds of sample fluid.

While the mobile phase can be comprised of one solvent only, it may alsobe mixed from plural solvents. Such mixing might be a low pressuremixing and provided upstream of the pump 20, so that the pump 20 alreadyreceives and pumps the mixed solvents as the mobile phase.Alternatively, the pump 20 might be comprised of plural individualpumping units, with plural of the pumping units each receiving andpumping a different solvent or mixture, so that the mixing of the mobilephase (as received by the separating device 30) occurs at high pressureand downstream of the pump 20 (or as part thereof). The composition(mixture) of the mobile phase may be kept constant over time, the socalled isocratic mode, or varied over time, the so called gradient mode.

A data processing unit 70, which can be a conventional PC orworkstation, might be coupled (as indicated by the dotted arrows) to oneor more of the devices in the liquid separation system 10 in order toreceive information and/or control operation. For example, the dataprocessing unit 70 might control operation of the pump 20 (for instancesetting control parameters) and receive therefrom information regardingthe actual working conditions (such as output pressure, flow rate, etc.at an outlet of the pump). The data processing unit 70 might alsocontrol operation of the solvent supply 25 (for instance setting thesolvent/s or solvent mixture to be supplied) and/or the degasser 27 (forinstance setting control parameters such as vacuum level) and mightreceive therefrom information regarding the actual working conditions(such as solvent composition supplied over time, flow rate, vacuumlevel, etc.). The data processing unit 70 might further controloperation of the sampling unit 40 (for instance controlling sampleinjection or synchronization sample injection with operating conditionsof the pump 20). The separating device 30 might also be controlled bythe data processing unit 70 (for instance selecting a specific flow pathor column, setting operation temperature, etc.), and send—inreturn—information (for instance operating conditions) to the dataprocessing unit 70. Accordingly, the detector 50 might be controlled bythe data processing unit 70 (for instance with respect to spectral orwavelength settings, setting time constants, start/stop dataacquisition), and send information (for instance about the detectedsample compounds) to the data processing unit 70. The data processingunit 70 might also control operation of the fractionating unit 60 (forinstance in conjunction with data received from the detector 50) andprovides data back.

Reference numeral 90 schematically illustrates a switchable valve whichis controllable for selectively enabling or disabling specific fluidicpaths within apparatus 10.

In the following, referring to FIG. 2 , a sample injector 200 for use ina fluid separation system 10 as described in FIG. 1 for separatingcomponents of a fluidic sample in a mobile phase according to anexemplary embodiment of the invention will be explained.

The sample injector 200 comprises a switchable valve 202 (whichcorresponds to reference numeral 90 in FIG. 1 ), a sample loop 204 influid communication with the valve 202 and configured for receiving thefluidic sample from a vial 230, a metering pump 206 in fluidcommunication with the sample loop 204 and configured for introducing ametered amount of the fluidic sample on the sample loop 204, and acontrol unit 208 (such as a microprocessor or a central processing unit,CPU) configured for controlling switching of the valve 202 to transferthe sample loop 204 between a low pressure state and a high pressurestate via an intermediate state, as will be described below in furtherdetail. Control unit 208 is further adapted for controlling the meteringdevice 206 to at least partially equilibrate, during the intermediatestate, a pressure difference in the sample loop 204 between the lowpressure state and the high pressure state. Thus, the metering device206 (metering pump) is configured to generate a high pressure (inopposite to conventional syringe pumps). This metering device 206 isarranged within the split loop 204. The split loop 204 can becompressed. The precompression may be performed up to a system pressureof the pump 20.

As can be derived from FIG. 2 to FIG. 4 , the switchable valve 202comprises two valve members which are rotatable with respect to oneanother. By rotating these two valve members along a rotation axis whichis perpendicular to the paper plane of FIG. 2 , a plurality of ports 216formed in one of the valve members and a plurality of oblong arcuategrooves 218 formed in the other one of the valve members can beselectively brought in or out of fluid communication with one another.Since the various ports 216 are connected to dedicated ones of fluidicchannels of the fluidic system as shown in FIG. 2 , automaticallyswitching the valve 202 under control of the control unit 208 may allowto operate the fluidic system 10 in different fluid communicationconfigurations. The valve 202 is configured as a six port high pressurevalve in the embodiment of FIG. 2 .

Fluid communication between the high pressure pump 20 and the separationcolumn 30 can be accomplished by an according switching state of thevalve 202. In such a fluidic path, a high pressure of for instance 100MPa may be present which may be generated by the high pressure pump 20.In contrast to this, the pressure state in the sample loop 204 may befor instance smaller than 0.1 MPa when introducing a sample into thesample loop 204. When this sample loaded on sample loop 204 is to beloaded on column 30, the pressure in sample loop 204 is also high, forinstance 100 MPa.

For the purpose of loading the sample on the sample loop 204, a needle224 may be driven out of a correspondingly shaped seat 226 using a drive228 so that the needle 224 can be immersed into vial 230 accommodating afluidic sample to be loaded onto the sample loop 204. A loop capillary240 is provided in the sample loop 204 for at least partiallyaccommodating the introduced sample.

In a further operation mode, the needle 224 may be immersed in a flushport 232. Waste containers 234, 236 may be provided for receiving awaste fluid which can be pumped through the fluidic channels shown inFIG. 2 . Furthermore, for flushing the fluidic system 200, fluid from aflush solvent vial 238 may be sucked by a peristaltic pump 250 and maybe pumped through corresponding channels of the fluidic system shown inFIG. 2 .

The metering device 206 is configured as a high pressure meteringdevice, i.e. as a metering device which is capable of providing apressure of up to 100 MPa in the sample loop 204 by correspondinglymoving a reciprocating piston 210 of the high pressure metering device206.

Before describing further details of the sample injector 200, some basicrecognitions of the present inventors will be summarized based on whichexemplary embodiments of the invention have been developed.

According to an exemplary embodiment, flow perturbances may be reducedand component lifetime of a HPLC autosampler may be increased by aprecompression and/or decompression of its loop volume.

HPLC injection system used for pressures above 60 MPa (for instance 120MPa) are conventionally faced with various problems. The volume withinthe split loop (in the embodiment of FIG. 2 , the split loop includesparticularly high pressure metering device 206, loop capillary 240,needle 224, needle seat 226, seat capillary 270) may be exposed to veryhigh pressures in a main pass position which is illustrated in FIG. 2 .Since liquids (mobile phase and sample) under such high pressures are nolonger incompressible, this loop volume is being compressed.

Furthermore, switching the injector valve 202 to a bypass position asshown in FIG. 4 conventionally leads to a very fast decompression of theloop volume because it gets connected to atmospheric pressure suddenly.This fast decompression generates a strong acceleration of the liquidwhich passes with high flow rates through the channels of the injectorvalve 202. This high flow rate (also called “water jetting”) may causedelamination of a coating on the valve stator due to cavitation anderosion on the polymeric valve rotor seal.

On the other hand does the pump 20 deliver flow while the valve 202switches to a main pass mode shown in FIG. 2 . During this time, thevalve channel is getting deconnected from the pump 20. The pump 20 ispumping against the closed channel which results in a pressure increase.

At the same time the column 30 gets deconnected from the pump 20 andflow is no longer delivered on top of the column 30. Concurrently thesystem after the column 30 is open and via detector cell connected to anatmospheric pressure. This may also cause the column pressure todecrease.

The above-mentioned problems of conventional systems which may beovercome by the embodiments shown in FIG. 2 to FIG. 9 have differentconsequences. Firstly, the jet stream generated during decompressioncauses damage on rotor seal and stator of the valve 202. This may resultin a reduced valve lifetime. Switching the valve 202 furthermore causespressure/flow disturbances (perturbances) like pressure peaks. This maylead to precision problems of flow rates, etc. The closed valve 202causes the column pressure to drop. The reconnected valve 202 on theother hand forwards the flow generated by the pump 20 via split loop tothe column 30. The pressure may be at reduced level. However, at thebeginning of this operation, the column pressure may be still higher asthe split loop pressure. In that case there is a possibility for areverse flow to develop. After this, the pressure starts equilibratingand the pump 20 delivers a positive flow towards the column 30. Thepressure peaks and the reverse flow may conventionally reduce thelifetime of a column 30.

Exemplary embodiments of the invention, for instance the systemsdescribed in FIG. 2 to FIG. 9 may overcome these conventional problemsby taking particularly the measures explained in the following. In orderto reduce the observed effects, a modified valve 202 and modifiedoperation procedures are provided. The modified valve 202 has flowchannels which are different in length (compare different lengths of thearcuate sections of the grooves 218 in FIG. 2 ) and the modifiedoperations include stops to provide an intermediate valve state in aninclined position (compare FIG. 3 ).

By clockwise turning the valve 202 from main pass (or start/inject)position as shown in FIG. 2 , the column 30 is connected to the pump 20via the split loop or sample loop 204. At the inclined position(pre/decompression mode as shown in FIG. 3 ), column 30 is connecteddirectly to the pump 20. In this inclined position, the split loop (i.e.loop capillary 240 plus metering device 206 plus needle 224 plus seatcapillary 270) is now isolated from the pump 20 and the column 30 but isstill under high pressure. In order to reduce that high pressure, piston210 of the metering device 206 can be drawn back a controlled amount forinstance until the loop pressure equals atmospheric pressure. Forinstance, this can be done by using a metering device as disclosed forinstance in EP 0,327,658 B1, U.S. Pat. No. 4,939,943 which allows highpressure applications.

With the loop pressure being brought close to atmospheric pressure, thevalve 202 can be again turned clockwise to its bypass position which isshown in FIG. 4 . This bypass position may also be denoted as a loadposition. Since there is no pressure gradient between the internal looppressure and the atmospheric pressure, no water jetting can develop.Therefore, both the delamination of the stator coating and the erosionof the polymer rotor may be eliminated or at least suppressed. Theresult is an increased lifetime of the valve 202 and of the entiresampling unit 200.

The valve 202 is in the bypass or load position in FIG. 4 , and theautosampler is ready to take a sample from vial 230. In a firstprocedure, the needle 224 may be lifted and moved into the sample vial230 or a well position (for instance of a multi-well plate). Now, thepiston 210 of the metering device 206 may be drawn back to a controlledpreset amount (for instance 2 .mu.l). Next, the needle 224 is seated inits seat 226, and the split loop 204 is closed thereby.

The valve 202 is then turned counterclockwise to the inclined positionshown in FIG. 3 where the pump 20 is still connected to the column 30.The split loop 204 is closed on both ends. If now the piston 210 of themetering device 206 is moved forward in a controlled manner, itsdisplacement generates a positive pressure and precompresses the trappedvolume. This pressure, potentially sensed by a pressure sensor 220, isbeing increased until it equals the system pressure.

This is the trigger to turn the valve 202 completely to the main passposition which is illustrated in FIG. 2 . Because the pressure of thesystem and the split loop 204 are equal at beginning of this operation,there will be only a very small pressure drop causing only minimum flowdisturbances. The pump 20 delivers the mobile phase through the splitloop 204 and pushes the sample onto the column 30 where thechromatographical separation of the sample may start.

Hence, FIG. 2 to FIG. 4 show schematically three positions of theinjection valve 202 of the autosampler 200 within HPLC system 10 duringthe injection cycle.

In the main pass position shown in FIG. 2 , a start or inject positionis shown where the rotor seal flow channels connect pump 20 with thesplit loop 204 and the seat capillary of the split loop 204 with theseparation column 30.

In the inclined position shown in FIG. 3 , the split loop volume getsdecompressed or precompressed.

In the bypass position shown in FIG. 4 , the flow channels of the rotorseal connect the pump 20 directly to the separation column 30 and thesplit loop 204 to the waste outlet 236.

Next, referring to FIG. 5 to FIG. 9 , a sample injector 500 in a liquidchromatography system 10 according to another embodiment of theinvention will be explained.

FIG. 5 illustrates a load or bypass position, FIG. 6 illustrates aprecompress position and FIG. 7 illustrates an inject position (or mainpass position) of the sample injector 500.

A main difference between the sample injector 500 and the sampleinjector 200 is the arrangement of the valve 502 which in an embodimentof FIG. 5 to FIG. 9 is configured as a multi-position/seven port highpressure valve.

Furthermore, in the embodiment of FIG. 5 to FIG. 9 , three differentflush solvent vials 238 are provided and three different flush ports 232are provided. Selection between three flush channels A, B and C can beperformed by correspondingly switching a low pressure selection valve504. Furthermore, a low pressure flush pump 506 is provided forperforming the flushing performance.

The multi-position valve 502 is provided for additionallyprecompressing, pump priming and pressure testing. All drawn sample getsinjected. Additional flush pump 506 may be for instance a syringe pumpfrom the company Tecan. Such an additional flush pump 506 may allowflushing of the sample loop 204 using the three flush ports A, B, C (forinstance two organic flush ports and one water flush port).

FIG. 8 illustrates the system of FIG. 5 to FIG. 7 in a prime pumpposition, and FIG. 9 illustrates the system 500 in a pressure testposition.

It should be noted that the term “comprising” does not exclude otherelements or features and the “a” or “an” does not exclude a plurality.Also elements described in association with different embodiments may becombined. It should also be noted that reference signs in the claimsshall not be construed as limiting the scope of the claims.

1. A method of injecting a sample volume into a chromatography column ofa chromatography system, the chromatography system comprising: a sampleloop in fluid communication with an injection valve, wherein the sampleloop comprises a sample conveying device for loading the sample volumein the sample loop, wherein the sample conveying device comprises a pumpvolume structure and a moveable element, wherein the moveable element isguidable within the pump volume structure; and a high-pressure fluidicpath in fluid communication with the chromatography column and ahigh-pressure pump, wherein the method comprising: flowing an eluentinto the high-pressure fluidic path at a pump pressure generated by thehigh-pressure pump; isolating the sample loop from the high-pressurefluidic path, wherein the isolating of the sample loop comprises placingthe injection valve in a PRESSURE COMPENSATION position; while thesample loop is isolated from the high-pressure fluidic path, sucking thesample volume into the sample loop from a sample vial by moving themoveable element with a stepping motor relative to the pump volumestructure; while the sample volume is loaded into the sample loop, andwhile the sample loop is isolated from the high-pressure fluidic path,moving the moveable element relative to the pump volume structure, apredetermined first distance, wherein the predetermined first distanceis based at least in part upon a compressibility of the eluent in thesample loop and the pump pressure; while the compressed sample volume iscompressed to the high pressure, connecting the sample loop to thehigh-pressure fluidic path; and while the compressed sample volume iscompressed to the high pressure, conveying the compressed sample volumefrom the sample loop to the chromatography column.
 2. The method ofclaim 1, wherein the sample loop includes a first connecting piece and asecond connecting piece, wherein the first connecting piece is connectedto a first sample loop port of the injection valve and to the sampleconveying device, wherein the second connecting piece is connected to asecond sample loop port of the injection valve and to the sampleconveying device, wherein the second connecting piece includes an intakesegment and a feed segment, wherein the intake segment and the feedsegment are configured to be separated.
 3. The method of claim 1,wherein in the PRESSURE COMPENSATION position, i) first and secondsample loop ports of the injection valve are closed so as to facilitatea pressurization of the sample loop, and ii) first and secondhigh-pressure ports of the injection valve are connected so as tooperatively connect the high-pressure pump in fluid communication withthe high-pressure fluidic path to the chromatography column, the methodfurther comprising: determining the compressibility of the eluent withthe high-pressure pump.
 4. The method of claim 1 further including:after the compressed sample volume has been conveyed from the sampleloop to the chromatography column, isolating the sample loop from thehigh-pressure fluidic path; and while the sample loop is isolated fromthe high-pressure fluidic path, moving the moveable element relative tothe pump volume structure, a predetermined second distance, to therebydecompress the sample loop to a pressure that essentially corresponds toan atmospheric pressure.
 5. The method of claim 1, wherein the moveableelement is connected to the stepping motor which is operable to move themoveable element within the pump volume structure, and the methodfurther comprises: measuring a force exerted upon the moveable elementby the stepping motor.
 6. The method of claim 1, wherein thecompressibility of the eluent and an elasticity of the sample loop arestored for use by the chromatography system.
 7. The method of claim 1,wherein the pump volume structure comprises a syringe and the moveableelement comprises a plunger.
 8. The method of claim 1, wherein thesample volume comprises the eluent, wherein the predetermined firstdistance is also based at least in part upon an elasticity of the sampleloop.
 9. The method of claim 1, wherein the stepping motor comprises anintegrated sensor measuring a force applied by the stepping motor on themoveable element.
 10. The method of claim 1 further comprising: afterthe compressed sample volume has been conveyed from the sample loop tothe chromatography column, isolating the sample loop from thehigh-pressure fluidic path; and while the sample loop is isolated fromthe high-pressure fluidic path, moving the moveable element relative tothe pump volume structure, a predetermined second distance, wherein thepredetermined second distance is based at least in part upon acompressibility of an eluent in the sample loop, to thereby decompressthe sample loop to a pressure that essentially corresponds to anatmospheric pressure.
 11. The method of claim 10, wherein thepredetermined second distance is also based at least in part upon thepump pressure.